Biologically active synthetic oligonucleotides, including aptamers, antisense oligonucleotides, ribozymes and, more recently, short interfering RNAs (siRNAs), have revolutionized molecular medicine by allowing the direct and specific targeting of individual genes and proteins. The ability to sensitively detect and quantify even minute quantities of such exogenous oligonucleotides in a biological sample will be a critical key to their development as clinically relevant therapeutics and diagnostics.
Aptamers (Gr. Aptus—to fit; meros—part or region) are oligonucleotides that bind with very high specificity and affinity to target molecules, including proteins. Aptamer binding relies on the specific three dimensional conformation of the properly folded oligonucleotide. Aptamers, can be generated against amino acids, drugs, proteins and other molecules. They can be isolated from combinatorial libraries of synthetic nucleic acid by an iterative process of adsorption, recovery, and reamplification. Following selection of a primary RNA oligonucleotide sequence that binds tightly to the target, a number of secondary modifications to the backbone, sugars, and bases, as well as to the 5′ and 3′ ends of the sequence, may be made in order to optimize the stability and/or antagonist (or agonist) biological activity of the aptamer. For example, aptamer oligonucleotides are frequently modified at 2′-ribo positions (e.g. with 2′-fluro or 2′-O-methyl) to make them more nuclease resistant. In addition, the 3′ ends of aptamers are frequently modified with an “inverted T cap” (i.e., addition of -3′dT at the 3′ end of the aptamer) to increase nuclease resistance, and the use of such an “inverted T cap” structure at the 5′ ends of aptamers has also been described (see, U.S. Appln. Ser. No. 60/493,500). Furthermore, the addition of non-immunogenic, high molecular weight or lipophilic compounds to the 5′ end, or other position, to improve nuclease resistance and/or other pharmacokinetic properties, has also been described (see, e.g., U.S. Pat. Nos. 6,011,020, 6,147,024, 6,229,002, 6,426,335, 6,465,188, and 6,582,918). Finally, the addition of soluble, high molecular weight steric groups, such as dextran or polyethylene glycol, to improve the antagonist properties of an aptamer has been described (see U.S. application Ser. No. 11/105,279).
An important problem encountered with the use of modified oligonucleotides in therapeutic applications is confirming the presence and persistence of the modified oligonucleotide in the subject. Although there have been numerous methods for detecting and/or quantifying nucleic acids in a biological sample, the presence of extensive modifications in oligonucleotide aptamers and certain antisense agents, in combination with their relatively short length, makes their detection and quantification difficult. Indeed, some high molecular weight modifications, such as 5′-PEGylation, interfere with certain conventional means of detection. Accordingly, a reliable, sensitive and quantitative method for detecting modified oligonucleotides present in a host sample would be very useful.